The present invention is directed to gene therapy vectors and methods, and provides a family of novel recombinant retroviral vectors capable of efficiently transferring any gene of interest into a wide range of mammalian target cells. Cells transduced with the recombinant retroviral vectors of the invention are capable of expressing high levels of a desired gene product for long periods of time. Thus, such transduced cells may be useful in the treatment of a wide variety of diseases wherein permanently augmenting or adding the production of a given protein or other polypeptide is therapeutically desirable. Preferred vectors of the invention lacking selectable markers are described, and are particularly useful for somatic cell gene therapy in the treatment of diseases wherein the co-production of marker gene products, such as antibiotics, would be undesirable or unacceptable.
Numerous methods exist for genetically engineering mammalian cells. There is great interest in genetically engineering mammalian cells for several reasons including the need to produce large quantities of various polypeptides and the need to correct various genetic defects in the cells. The methods differed dramatically from one another with respect to such factors as efficiency, level of expression of foreign genes, and the efficiency of the entire genetic engineering process.
One method of genetically engineering mammalian cells that has proven to be particularly useful is by means of retroviral vectors. Retrovirus vectors and their uses are described in many publications including Mann, et al., Cell 33:153-159 (1983) and Cone and Mulligan, Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984). Retroviral vectors are produced by genetically manipulating retroviruses.
Retroviruses are RNA viruses; that is, the viral genome is RNA. This genomic RNA is, however, reverse transcribed into a DNA copy which is integrated stably and efficiently into the chromosomal DNA of transduced cells. This stably integrated DNA copy is referred to as a provirus and is inherited by daughter cells as any other gene. As shown in FIG. 1, the wild type retroviral genome and the proviral DNA have three Psi genes: the gag, the pol and the env genes, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins. The 5xe2x80x2 and 3xe2x80x2 LTRs serve to promote transcription and polyadenylation of virion RNAS.
Adjacent to the 5xe2x80x2 LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). Mulligan, R.C., In: Experimental Manipulation of Gene Expression, M. Inouye (ed), 155-173 (1983); Mann, R., et al., Cell, 33:153-159 (1983); Cone, R. D. and R. C. Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984).
If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Mulligan and coworkers have described retroviral genomes from which these Psi sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome. Mulligan, R. C., In Experimental Manipulation of Gene Expression, M. Inouye (ed), 155-173 (1983); Mann, R., et al., Cell, 33:153-159 (1983); Cone, R. D. and R. C. Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984). Additional details on available retrovirus vectors and their uses can be found in patents and patent publications including European Patent Application EPA 0 178 220, U.S. Pat. No. 4,405,712, Gilboa, Biotechniques 4:504-512 (1986) (which describes the N2 retroviral vector). The teachings of these patents and publications are incorporated herein by reference.
Retroviral vectors are particularly useful for modifying mammalian cells because of the high efficiency with which the retroviral vectors xe2x80x9cinfectxe2x80x9d target cells and integrate into the target cell genome. Additionally, retroviral vectors are highly useful because the vectors may be based on retroviruses that are capable of infecting mammalian cells from a wide variety of species and tissues.
The ability of retroviral vectors to insert into the genome of mammalian cells have made them particularly promising candidates for use in the genetic therapy of genetic diseases in humans and animals. Genetic therapy typically involves (1) adding new genetic material to patient cell in vivo, or (2) removing patient cells from the body, adding new genetic material to the cells and reintroducing them into the body, i.e., in vitro gene therapy. Discussions of how to perform gene therapy in a variety of cells using retroviral vectors can be found, for example, in U.S. Pat. No. 4,868,116, issued Sep. 19, 1989, and U.S. Pat. No. 4,980,286, issued Dec. 25, 1990 (epithelial cells), WO89/07136 published Aug. 10, 1989 (hepatocyte cells) , EP 378,576 published Jul. 25, 1990 (fibroblast cells), and WO89/05345 published Jun. 15, 1989 and WO/90/06997, published Jun. 28, 1990 (endothelial cells), the disclosures of which are incorporated herein by reference.
In order to be useful for the various techniques of gene therapy, suitable retroviral vectors require special characteristics that have not hitherto been available. A primary source of the need for these special requirements of the vector for use in the in vivo genetic manipulation of patient cells in gene therapy is because it is usually not feasible to use retroviral vectors that require a selection for integration of the vector into the genome of xe2x80x9cpatientxe2x80x9d cells. For example, typical retroviral vectors, e.g., MSV DHFR-NEO described in Williams, et al., Nature 310:476-480 (1984), use neomycin resistance as a suitable marker for detecting genetically modified cells. Thus, with such neomycin resistant retroviral vectors, patients would be required to be exposed to high levels of neomycin in order to effect genetic repair of cells through in vivo gene therapy. Moreover, in both in vivo and in vitro gene therapy it may be undesirable to produce the gene product of the marker gene in cells undergoing human gene somatic therapy. For example, there is no therapeutic reason to produce large levels of neomycin phosphotransferase in blood cells undergoing hemoglobin gene replacement for curing a thalassemia. Therefore, it would be desirable to develop retroviral vectors that integrate efficiently into the genome, express desired levels of the gene product of interest, and are produced in high titers without the coproduction or expression of marker products such as antibodies.
Despite considerable progress in efforts to develop effective genetic therapies for diseases involving hematopoietic cells, a number of significant technical hurdles remain. First, while a variety of transduction protocols have been developed which make it possible to efficiently transfer genes into murine hematopoietic stem cells, it has not yet been possible to achieve efficient gene transfer into reconstituting cells of large animals. It is currently unclear to what extent this problem is vector related (e.g. insufficient titers, host range) or a consequence of a lack of knowledge regarding the optimal conditions for obtaining the proliferation and/or efficient engraftment of appropriate target cells. A second important technical stumbling block relates to the development of retroviral vectors possessing the appropriate signals for obtaining high level constitutive expression of inserted genes in hematopoietic cells in vivo. Although a number of groups have demonstrated the expression of genes in mice reconstituted with transduced bone marrow cells, others have experienced difficulties (10-12). Overall, few general principles regarding features of vector design important for gene expression in vivo have emerged. In particular, because of differences in vector backbones, inserted genes, viral titers, transduction protocols, and other experimental parameters, it has been impossible to directly compare the performance of different vectors and to determine the features of vector design which most critically affect gene expression in hematopoietic cells in vivo. In addition, few studies have examined the ability of transferred genes to be expressed for very long periods of time (e.g. the lifetime of the transplant recipients), a clearly important goal of gene therapy for diseases involving hematopoietic cells.
The present invention is directed to a family of novel retroviral vectors capable of being used in somatic gene therapy. The retroviral vectors of the invention include an insertion site for a gene of interest and are capable of expressing desired levels of the encoded protein in a wide variety of transfected cell types.
In one aspect of the invention there is provided a retroviral vector comprising in operable combination, a 5xe2x80x2 LTR and a 3xe2x80x2 LTR derived from a retrovirus of interest, and an insertion site for a gene of interest, and wherein at least one of the gag, env or pol genes in the vector are incomplete or defective. The vector preferably contains a splice donor site and a splice acceptor site, wherein the splice acceptor site is located upstream from the site where the gene of interest is inserted. Also, the vector desirably contains a gag transcriptional promoter functionally positioned such that a transcript of a nucleotide sequence inserted into the insertion site is produced, and wherein the transcript comprises the gag 5xe2x80x2 untranslated region. The preferred vectors of the invention are lacking a selectable marker, thus, rendering them more desirable in human somatic gene therapy because a marker gene product, such as an antibiotic drug marker, will not be co-produced or co-expressed.
The gene of interest that is incorporated in the vectors of the invention may be any gene which produces a hormone, an enzyme, a receptor or a drug(s) of interest.
The retroviral vectors are most suitably used in combination with certain packaging cells, as herein defined, which in turn may be used in a wide variety of cell types for human or animal somatic gene therapy.
A particular preferred retroviral vector of the invention is identified herein as xe2x80x9cMFGxe2x80x9d, as depicted in FIGS. 2c and 3, and the plasmid containing it, and especially the plasmid MFG having the identifying characteristics of ATCC No. 68,754.
The present invention is also directed to retroviral vectors similar to those described above, but further comprising a non-LTR enhancer and the alpha-globin transcriptional promoter sequence in order to control the expression of various genes of interest. This aspect of the invention specifically provides for the use of an enhancer sequence from cytomegalovirus. Also provided are vectors in which the enhancer sequence is deleted from the 3xe2x80x2 LTR thus resulting in the inactivation of the 5xe2x80x2 LTR upon integration of the vector into the genome. The xcex1-globin promoter containing vector xcex1-SGC is specifically provided, and especially that which is depicted in FIG. 4, and the plasmid containing it, and especially the plasmid xcex1-SGC having the identifying characteristics of ATCC No. 68,755.